The functional capacity of genetically-encoded proteins is powerfully expanded by reversible posttranslational acetylation. Our long term goal is to understand the catalytic mechanism and substrate specificity of protein deacetylases, the enzymes which hydrolyze acetyl groups from the epsilon-NH2 side chain of lysyl residues within histone N-termini as well as proteins such as p53 and HIV-Tat. Protein deacetylases play a critical role in transcriptional repression, chromatin remodeling, and modulation of activities of oncogenic proteins, transcription factors, and mitotic proteins. Inhibitors of these enzymes are powerful anticancer agents that bring about morphological changes in cancer cells, reverting transformed cells to a normal-like phenotype by stimulating cellular differentiation and cell cycle machinery. Understanding the mechanisms, specificity, and global function of these important proteins will have pronounced impact on our ability to eradicate human cancer and better our understanding of the complex biology of transcription. In this proposal we seek to gain a complete view of histone deacetylase mechanism, specificity, and inhibition. In Aim 1 we will rigorously characterize the catalytic amidohydrolase mechanism of Hos3 from S. cerevisiae, a prototypical Class I/II zinc-dependent histone deacetylase. In Aim 2, analysis of the zinc-dependent enzymes will be complemented by a full characterization of S. cerevisiae Hst2, a prototypical member of the newly discovered NAD+-dependent (Sir2- like) Class III histone deacetylases. Finally, in Aim 3 we will examine the substrate specificity of histone deacetylases using well defined site-specifically modified peptides, full-length histones, and compositionally defined synthetic nucleosome assemblies. The effects of microsequence context, posttranslational modification state/position, partner protein and DNA directive groups, isoform selectivity, and histone/nucleosome composition and conformation on deacetylase specificity will be determined. Together, these studies will provide a clear picture of protein deacetylase mechanism, inhibition, and substrate specificity, and will direct the development of mechanism-based, isoform-selective inhibitors.